It has become increasingly important to determine the relative abundance of protein or endogenous peptide expression levels in different biological states using mass spectrometry (MS). MS is a technique growing in popularity in proteomics due, in part, to its ability to detect the presence of various chemical moieties such as metabolites, proteins, and nucleic acids. MS can provide both qualitative and quantitative information about the molecule provided that a proper internal standard or marker is used.
Numerous MS-based chemical derivatization quantitation approaches such as mass-difference labeling and isobaric labeling methodologies have been developed and widely used for quantitative proteomics and peptidomics (Ong et al., Nat. Chem. Biol. 2005, 1:252-262). Mass-difference labeling approaches introduce a mass difference for the same peptide by incorporating a light or heavy isotopic form of the labeling reagent. Light and heavy labeled peptides are combined prior to MS analysis, and quantitation is accomplished by comparing the extracted ion chromatogram peak areas of light and heavy forms of the same peptide. Methods such as isotope-coded affinity tags (ICAT), stable isotope labeling with amino acids in cell culture (SILAC), 4-trimethylammonium-butyryl (TMAB) labels, and reductive formaldehyde dimethylation have been widely used in mass-difference quantitation proteomics (Gygi et al., Nat. Biotechnol. 1999, 17:994; Li et al., Mol. Cell. Proteomics 2003, 2:1198-1204; Hansen et al., Mol. Cell. Proteomics 2003, 2:299-314; Ong et al., Mol. Cell. Proteomics 2002, 1:376-386; Zhang et al., Anal. Chem. 2002, 74:3662-3669; and Hsu et al., Anal. Chem. 2003, 75:6843-6852).
Although being well-established methodologies for quantitative proteomics, mass-difference labeling has two general limitations. First, only a binary set of samples can be compared due to the use of light and heavy labeling of a peptide. Second, mass-difference labeling increases mass spectral complexity by introducing an extra pair of labeled peptides, thus decreasing the confidence and accuracy of quantitation. The first limitation has been addressed and overcome by several research groups by introducing multiple heavy labeled reagents, rather than just one (Hsu et al., Electrophoresis 2006, 27:3652-3660; Morano et al., Anal. Chem. 2008, 80:9298-9309; and Boersema et al., Proteomics 2008, 8:4624-4632). However, the second limitation is an inherent drawback of the mass-difference approach, and the spectral complexity is only increased with the use of multiple heavy isotope labeling reagents.
These limitations can be addressed through the use of isobaric labeling. There are two popular brands of isotopic labeling reagents, iTRAQ and TMT, currently sold in order to tag peptides when performing quantitative analysis of peptides using MS. Tandem mass tags (TMTs) were the first isobaric labeling reagents used to improve the accuracy for peptide and protein quantitation by simultaneous identification and relative quantitation during tandem mass spectrometry (MS/MS or MS2) experiments (Thompson et al., Anal. Chem. 2003, 75:1895-1904). Two generations of TMTs were reported (TMT1 and TMT2), and each generation had two isobaric labels. Amine groups (N-terminus and ε-amino group of the lysine side chain) in peptides labeled with TMT1 produce fragments at m/z 270 and 273 at 70 V collision energy, whereas TMT2 produces fragments at m/z 287 and 290 at 35 V collision energy. Relative quantitation can be performed by comparing the intensities of these fragments to one another. A 6-plex version of TMTs was also recently reported (Dayon et al., Anal. Chem. 2008, 80:2921-2931).
iTRAQ follows the same principle as TMTs quantitation, but it improves the quantitation further by providing four isobaric labels with signature reporter ions that are one Da apart upon MS2 fragmentation (Ross et al., Mol. Cell. Proteomics 2004, 3:1154-1169). Thus, iTRAQ allows for the quantitation of proteins present in four different biological states simultaneously (a 4-plex quantitation). These tags are structurally identical isobaric compounds with different isotopic combinations. Each sample is labeled individually, pooled together, and introduced into the mass spectrometer for quantitative analysis. Since samples are isobarically labeled, the same peptide from four samples produces a single peak in MS mode, but upon MS2 fragmentation, each labeled sample gives rise to a unique reporter ion (m/z 114.1, 115.1, 116.1, and 117.1) along with sequence-specific backbone cleavage for identification. Relative quantitation is achieved by correlating the relative abundance of each reporter ion with its originating sample.
iTRAQ 8-plex quantitation follows the same quantitation principle as iTRAQ 4-plex quantitation (C. Leila, et al., Proteomics 2007, 7. 3651-3660). Instead of using four reporter ions (m/z 114.1, 115.1, 116.1, and 117.1) for quantitation of four samples, eight reporter ions (m/z 114.1, 115.1, 116.1, 117.1, 118.1, 119.1, 121.1 and 122.1) can be produced and used for simultaneous quantitation of eight samples. iTRAQ 8-plex reagents double the quantitation throughput over the 4-plex reagents. In addition to the higher throughput over a wider quantitation dynamic range, 8-plex reagents can also provide more accurate quantitation.
A common problem with the 4-plex iTRAQ reagent is that because the four reporter ions are only one Dalton apart, isotope peak gains from or losses to adjacent reporter ions affect both the accuracy and dynamic range of quantitation for 4-plex samples. The quantitation accuracy problem can be overcome by employing a complicated mathematic algorithm to quantify the reporter ions (A. Boehm, et al., BMC Bioinformatics 2007, 8. 214). A software package is needed for 4-plex quantitation which brings extra cost for data analysis. The mathematic approach works well for quantifying samples within ten-fold ratio difference. However, if two samples labeled by two adjacent reporter ions have abundance difference greater than ten-fold, the reporter ion representing lower concentration sample could potentially be buried by the isotope peak of the adjacent reporter ion representing high concentration sample (S. Y. Ow, et al., J. Proteome Res. 2009, 8. 5347-5355). Therefore, the quantitation dynamic range is reduced. Two adjacent reporter ions with one Dalton mass difference should be avoided to quantify samples varying in concentration greater than ten-fold. In this situation, 4-plex reagents can only be used to quantify two samples. 8-plex reagents can provide two Dalton mass difference reporter ions for quantitation of four samples. Because of the minimal interference of adjacent reporter ions, accurate quantitation and wider quantitation dynamic range can be achieved for four samples without sophisticated mathematical processing. Demand of high throughput protein/peptide LC/MS/MS quantitation in practice makes iTRAQ 8-plex highly desirable for multiple sample quantitation. However, the trade-off of accurate quantitation and wider dynamic range is the high price of iTRAQ 8-plex reagents (about $2,500 for a kit for five trials).
Isobaric MS2 tagging approaches have also been successfully used in MS-based quantitative proteomics. However, their application as a routine tool for quantitative MS studies is limited by high cost. The high cost of commercial TMTs and iTRAQ comes from the challenge of synthesizing these compounds as multiple steps involved in the synthesis lead to moderate to low yields. A set of 6-plex deuterium-labeled DiART reagents was reported very recently with reduced cost of isobaric labeling. However, seven steps were still required to synthesize these compounds with only 30%-40% overall yield (Zeng et al., Chem. Commun. 2009, 3369-3371). Additionally, many alternate MS labels are too labile which leads to cleaving the tag from the peptide of interest during mass spectrometry analysis.
A new type of isobaric MS2 tags with fewer steps involved in synthesis is desirable to further reduce experimental cost while taking full technical advantages of the isobaric MS2 tagging approach. Formaldehyde dimethylation represents one of the most affordable approaches among all isotopic chemical derivatization techniques used for MS-based peptide and protein quantitation (Boersema et al., Proteomics 2008, 8:4624-4632; Ji et al., Proteome Res. 2005, 4:2099-2108; Ji et al., Proteome Res. 2005, 4:1419-1426; Ji et al., Proteome Res. 2005, 4:734-742; Huang et al., Proteomics 2006, 6:1722-1734; Ji et al., Proteome Res. 2006, 5:2567-2576; Ji et al., Anal. Chim. Acta 2007, 585:219-226; Guo et al., Anal. Chem. 2007, 79:8631-8638; Wang et al., J. Proteome Res. 2009, 8:3403-3414; Raijmakers et al., Mol. Cell. Proteomics 2008, 7:1755-1762; Synowsky et al., J. Mol. Biol. 2009, 385:1300-1313; Lemeer et al., Mol. Cell. Proteomics 2008, 7:2176-2187; Khidekel et al., Nat. Chem. Biol. 2007, 3:339-348; Rogers et al., Proc. Natl. Acad. Sci. U.S.A. 2007, 104:18520-18525; Aye et al., Mol. Cell. Proteomics 2009, 8:1016-1028; and Boersema et al., Nat. Protocols 2009, 4:484-494). However, isotopic formaldehyde labeling is a mass-difference labeling approach and, thus, lacks the advantages offered by the isobaric labeling approach.
The present inventors previously developed and demonstrated a set of novel and cost effective N, N-dimethylated leucine (DiLeu) 4-plex reagents as an attractive alternative to iTRAQ reagent for protein and peptide quantitation (F. Xiang, et al., Anal. Chem. 2010, 82. 2817-2825). However, there is no alternative to 8-plex iTRAQ reagents currently on the market. Therefore, what is needed are improved isobaric tandem MS tags that are simple to synthesize, highly efficient in labeling small molecules and peptides, more stable than the current commercial labeling reagents, and are significantly easier and less expensive to produce. Developing a set of novel 8-plex, or even 16-plex, reagents with greatly reduced experimental cost would be beneficial for increasing the throughput of multiple sample quantitation and achieving accurate quantitation of multiple samples in a routine manner.